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Organic acidemias MSUD

1. Major phenotypic expression:

Overwhelming illness in the first days of life with lethargy progressive tocoma, opisthotonus, and convulsions; recurrent episodes leading to developmental delay; characteristic maple syrup odor, branched-chain amino acidemia andaminoaciduria; branched-chain oxoaciduria; deficiency of branched-chain oxoaciddehydrogenase.

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MSUD - introduction

accumulation in body fluids elevated quantities of leucine, isoleucine, and

valine and their corresponding oxoacids unusual odor quite like that of maple syrup

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Metabolic pathways in the catabolism of leucine, isoleucine and valine. The

site of the defect is shown at the oxo-acid step in each of the three pathways.

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MSUD clinical features

Infants with classic MSUD - normal at birth, but remain well for only a fewdays Vomiting or difficulty to feed - early symptoms By the end of the first week - lethargic, progressive neurologicdeterioration periods of flaccidity alternating with hypertonicity abnormal eye movements, convulsions occur regularly Finally apnea, coma, and death

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MSUD genetics and pathogenesis

an autosomal recessive trait

all ethnic groups The fundamental defect is in the activity of the branchedchain oxoaciddehydrogenase multienzyme complex: E1- a decarboxylase; E2 - an acyltransferase; E3 - a flavoprotein lipoamide dehydrogenase enzyme activity can be measured in human liver, kidney and leukocytes,cultured fibroblasts or lymphoblasts and amniotic fluid cells mutations scanning with allele-specific oligonucleotide probes

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MSUD - genetics

The E1 gene chromosome 19q13.1-13.2

E1Beta - 6p21-22E2 - 1p31E3 - 7q31-32Mutations occur in each gene, but a majority in the E1 and E2 genes.The mutation in the Mennonite population (MSUD is common) is a T to Atransition - yields a single missense tyrosine to asparagine change atposition 393 (Y393N)

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MSUD - screening

Rapid screening for MSUD - tandem MS - forms the basis for all of theneonatal screening programs for this disease.

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Amino acid metabolism disorders - Phenylketonuria

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What is it?

autosomal recessive disorder (commonly known as PKU)

increases the levels of an aminoacid called phenylalanine in the bloodphenylalanine is an indispensable amino acid obtained through dietIf not treated, phenylalanine builds up to harmful levels in the body, causingintellectual disability and other serious health problems.

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What are the symptoms?

the signs and symptoms vary from mild to severe.classic PKU - most severe formwithout treatment development of permanent intellectual disability after fewmonthscommon: seizures, delayed development, behavioral problems, and psychiatricdisordersmusty or mouse-like odor - side effect of excess phenylalanine in the body.lighter skin and hair than unaffected family membersskin disorders - eczema.less severe forms - variant PKU and non-PKU hyperphenylalaninemiasmaller risk of brain damage.treatment not required

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PregnancyBabies born to mothers with PKU and uncontrolled phenylalanine levels significant risk of intellectual disability due to exposure to very high levels ofphenylalanine before birthlow birth weightgrow slowlyheart defects or other heart problemssmall head size (microcephaly)behavioral problemsrisk of pregnancy loss.

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How common is it?

The occurrence of PKU varies among ethnic groups and geographic regionsworldwideIn the United States, PKU occurs in 1 in 10,000 to 15,000 newborns.In Poland 1 in 8000 newbornsMost cases of PKU are detected shortly after birth by newborn screening, andtreatment is started promptly result: the severe signs and symptoms of classicPKU are rarely seen.

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Testing

Newborn screening: PAH deficiency is most commonly diagnosed upon routine screening of newborns. PAH deficiency can be detected in virtually 100% of cases by newborn screeningutilizing the Guthrie card bloodspot obtained from a heel prick.

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Testing

Three methods of newborn screening currently in use:

Guthrie card bacterial inhibition assay (BIA), a time-tested, inexpensive,simple, and reliable test Fluorometric analysis, a reliable quantitative and automated test which producesfewer false positive test results than the BIA Tandem mass spectrometry (MS), the same benefits as fluorometric analysis,can also measure tyrosine concentration, and be useful in interpreting Pheconcentration. Can be used to identify numerous other metabolic disorders on thesame sample

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Clinical testing

Targeted mutation analysis.

A panel of four to 15 common point mutations and very small deletions -detectionrate of approximately 30%-50%. Mutation scanning.detects virtually all point mutations in the PAH gene. Mutation scanning by DHPLC a fast and very efficient method to detect locus-specific point mutations Sequence analysis.Sequencing of all 13 exons - mutation detection rate of about 99%. Duplication/deletion analysis.Comparative multiplex dosage analysis - useful in detecting large duplications ordeletions when no mutations have been identified by mutation scanning or sequenceanalysis.

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Prevalence

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Treatment of Manifestations Restriction of dietary phenylalanine.normalization of the concentrations of Phe and Tyr in the bloodPhe concentrations of 120-360 mol/L (2-6 mg/dL) or 40-240 mol/L (1-4 mg/dL) regarded as safe.A diet restricted in Phe - initiated as soon as possible after birth and continued atleast into adolescenceThe diet must be carefully monitored so that growth and nutritional status areunaffected Supplementation with BH4.many individuals with PAH deficiency - responsive to the 6R-BH4 stereoisomer inpharmacologic doses (20 mg/kg daily in divided oral doses)the 6R-BH4 enhances in vivo phenylalanine hydroxylation and lowers plasmaphenylalanine concentration with improved tolerance of dietary phenylalanine

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Genetic counseling autosomal recessive disease at conception - sib of an affected individual:25% chance of being affected50% chance of being an asymptomatic carrier25% chance of being unaffected and not a carrier

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Molecular Genetic Testing

HGD - the gene encoding homogentisate 1,2-dioxygenase

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Normal allelic variants

The normal HGD gene3q21-q2354.3 kb14 exons1715-bp transcript

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Pathologic allelic variants

67 mutations in HGD have been reported The mutations are distributed throughout the HGD gene sequence. The majority of mutations: missense, but nonsense, frame shift, and splice-sitemutations do occur

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Normal gene product

homogentisate 1,2-dioxygenase - an enzyme in the phenylalanine/tyrosinedegradation pathway 445 amino acids expressed in the liver and kidney, small intestine, colon and prostate Homogentisate 1,2-dioxygenase functions in the metabolism of HGA by catalyzing anoxidative cleavage of the benzene ring to yield maleylacetoacetic acid. It requiresoxygen, ferrous iron, and sulfhydryl groups

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MCAD most common disorder of fatty acid oxidation 1 in 6000 to 10 000 caucasian births

2 general types of presentation:

The first - hypoketotic hypoglycemia is the clinical picture of Reyesyndrome The second - chronic disruption of muscle function with symptoms relevantto myopathy or cardiomyopathy, including weakness, hypotonia,congestive heart failure, or arrhythmia Another presentation is with the sudden infant death syndrome (SIDS)

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MCAD - genetics

MCAD deficiency is autosomal recessive

The gene is on chromosome 1 - ACADM A single mutation from A to G (985), leading to a lysine (K) to glutamic acid(E) change in residue 329 of the protein, accounts for virtually all of thepatients studied.

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MCAD - Prevalence

MCAD deficiency is prevalent in individuals of European (especially

northern) descent. The overall frequency of the disorder is between 1:4,900 and 1:17,000;Based on newborn screening programs worldwide, the incidence of MCADdeficiency has been defined in:Northern Germany (1:4,900 newborns)Southern Germany (1:8,500 newborns)New South Wales, Australia (1:25,000 newborns)USA (1:15,700 newborns)Taiwan (~1:700,000 live births)Japan (1:51,000 live births)

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Carbohydrate metabolism - galactosemia

MAJOR PHENOTYPIC EXPRESSION

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Galactose-1-phosphate uridyltransferase, the site of the enzymedefect in patients with galactosemia.The brackets indicate the uridyl andglucose-1-phosphate moieties ofUDPG which have split at the arrow inthe uridyl transferase reaction,transferring the uridyl group from G-1-Pof UDPG to galactose-1-phosphate(Gal-1-P) to formuridinediphosphogalactose(UDPGal).

Galactosemia - clinical manifestations

appear within days of birth or of the initiation of milk feedings - increase in

severity in the first months of life vomiting and jaundice - develop a few days after milk feedings are begun anorexia, failure to gain weight or to increase in length, or even weight loss hepatomegaly - constant finding on examination edema, ascites hypoprothrombinemia and bleeding. splenomegaly fatal - if milk feedings are continued

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Galactosemia - sepsis

sepsis neonatorum - most commonly E. coli

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Galactosemia - genetics

autosomal recessive trait

the enzyme defect - uridyl transferase - can be detected in the erythrocyteor in cultured fibroblasts and amniotic cells, leukocytes and liver in patients with classic galactosemia, the activity of the enzyme is virtuallycompletely absent Incidence rates 1:55000 (Duarte variant 1 in 3000-4000 but no clinicalmanifestations) 9p13 transferase gene (GALT) cDNA 3.9kb neonatal screening in the US

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Galactosemia most common mutations

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Protocol for galactosemia

Galactose

Galactose structure is identical to that of glucose except for the position of

the hydroxyl on carbon 4 Lactose - the principal sugar of mammalian milks - predominant dietarysource of galactose; it is a disaccharide in which glucose and galactose arelinked in an -1,4-glucosidic bond in which an oxygen bridge connectscarbon 1 of galactose and carbon 4 of glucose The pathogenesis of most of the clinical manifestations of galactosemia isthe accumulation of Gal-1-P in tissues Treatment exclusion of galactose from the diet

Hypoxanthine-guanine phosphoribosyl transferase

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Lesch-Nyhan disease

male infants - normal at birth, develop normally for the first 6 to 8 monthsfirst sign - orange crystals or orange sand in the diapers (crystalluria)defective motor development - evident in the second six months of lifefailure to reach developmental milestonespatients do not learn to walk, and must have some support even to situnaided but do learn to sit in a chair if fastened securely involuntary movements - 100 percent of patients self-injurious behaviour

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Diagnosis/testing demonstration of absent to near-absent beta-hexosaminidase A (HEX A) enzymaticactivity in the serum or white blood cells of a symptomatic individual in the presenceof normal or elevated activity of the beta-hexosaminidase B (HEX B) isoenzyme Mutation analysis of the HEXA gene: for genetic counseling purposes to1) to distinguish pseudodeficiency alleles from disease-causing alleles in individualswith apparent deficiency of HEX A enzymatic activity2) to identify specific disease-causing alleles in affected individuals

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Genetic counseling autosomal recessive manner At conception, each sib of an affected individual:25% chance of being affected50% chance of being an asymptomatic carrier25% chance of being unaffected and not a carrier

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Prevention of Tay-Sachs disease (19711992, showing 90%

reduction in the disease in Jewish population (19701993)

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Prevalence previously one in 3600 Ashkenazi Jewish births carrier rate for TSD: one in 30 among Jewish Americans of Ashkenazi extraction(Central and Eastern Europe). now the incidence of TSD in the Ashkenazi Jewish population - reduced by greaterthan 90% - result of extensive genetic counseling of carriers identified throughcarrier screening programs and monitoring of at-risk pregnancies Among Sephardic Jews and all non-Jews - 100 times less common - carrierfrequency (between 1/250 and 1/300) all ethnic, racial, and religious groups. Certain populations isolated genetically: French Canadians of Quebec, Cajuns fromLouisiana and the Old Order Amish in Pennsylvania - carry HEXA mutations withfrequencies comparable to or even greater than Ashkenazi Jews.

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Molecular genetics

Gene name: HEXA

Gene locus: 15q23-q24 Protein name: beta-hexosaminidase subunit alpha

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Normal allelic variants

35,000 bp contains 14 exons

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Pathologic allelic variants

more than 100 HEXA mutations identified to date >90 - acute infantile phenotype (Tay-Sachs disease) small insertions, deletions producing frameshifts, nucleotide substitutions producingstop codons Among Ashkenazi Jewish people - two mutations associated with the acute infantileform account for 90-95% of all alleles In the non-Jewish general population - two other mutations account for 35% allelesassociated with the acute infantile phenotype The mutations in Ashkenazi Jew:null alleles - no protein productgene is transcriptionally activeThe most frequent allele: 4-bp insertion in exon 11 - frameshift and downstreamstop codon in the coding sequence normal transcription - undetectable mRNAThe second major allele - donor splice-junction mutation in intron 12 - production ofseveral aberrantly spliced mRNAs.Zakad Genetyki Medycznej

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Abnormal gene product

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Cystic fibrosis

recognised 1936accumulation of thick mucous secretionsblockage of the airwayssecondary infectionsignificant cause of chronic ill health and mortality in childhood and early adult life.Icidence rate - 1 in 2000 to 1 in 3000African-Americans (1 in 15000)Asian-Americans (1 in 31 000).

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Diagnosis/testing diagnosis of cystic fibrosis - established in individuals with one or more characteristicphenotypic features of CF abnormality in CFTR function:presence of two disease-causing mutations in the CFTR geneabnormal sweat chloride values (>60 mEq/L)transepithelial nasal potential difference (NPD) measurements characteristic of CF